The combination of applied hydrostatic high pressure with polarized steady state fluorescence spectroscopy can provide important insights into altered conformation, dynamics and interactions of complex biological macromolecules in solution (See reference 1, infra.). Due to the non-compressibility of the aqueous solvent, applied pressure effects on the observed fluorescence emission anisotropy reflect exclusive alteration in the hydrodynamic volume of the system under investigation. Hence, protein conformations (See reference 1–4, infra.), dissociation and association of oligomeric proteins (See references 1,5,6, infra.), and altered lipid membrane structure (See reference 1,7–9 infra.) and/or dynamics (See references 10–12, infra.), can be readily studied at concentration levels of non infinite dilution. In addition, the technique can provide information regarding local flexibility or overall rotational dynamics of a system, depending on the nature of the fluorophore studied (See reference 1, 2, 4, 13, infra.).
However, a severe limitation of this approach is the inherent scrambling of the polarized light by the induced birefringence of the optical windows (quartz or to a lesser extent, sapphire) of the spectroscopy cell when pressures of greater than 0.2 kbar are applied. At pressures greater than 1 kbar, this so-called “scrambling” effect can be on the order of the measured fluorescence anisotropy. As a result, measured polarized fluorescence intensities are contaminated by scrambling artifacts, and determined values of the fluorescence emission anisotropy (EA) are significantly distorted.
In this regard, several approaches have been adopted for correction of measured polarized fluorescence pressure data. Paladini and Weber (See reference 14, infra.), using a well-characterized rotationally immobile fluorophore in glycerol at low temperatures, determined values for the scrambling correction factor (α(p)) as a function of increasing hydrostatic pressure, under the same optical conditions (i.e. excitation and emission wavelengths) as for the fluorophore of interest. Since the probe is rotationally restricted, deviations of <r> from that measured at zero pressure value directly reflect the combined depolarizing artifacts comprising scrambling effects of the optical windows and possible internal light reflections within the high pressure spectroscopy cell. Once scrambling factors have been determined, values of <r> for the measured system at any pressure can now be corrected. This method whilst effective, necessitates a separate experiment using a standard fluorophore system in order to determine values for the scrambling factors, α(p). Additionally, due to aging of the optical windows of the high pressure cell with applied hydrostatic pressure, values for α(p) can change between experiments, and should strictly be recorded for each experiment performed.
An alternate mechanical approach is to exclude possible scrambling artifacts by mounting the excitation and emission polarizers between the optical windows of the bomb and the sample cuvette, inside the high pressure spectroscopy cell (See reference 15, infra.). However, this approach is experimentally challenging as the polarizing material must be sandwiched between quartz plates, and sealed to exclude possible deleterious effects of the pressure transducing fluid (usually ethanol). Additionally, unless a rotating polarizer with remote access can be incorporated within the high pressure spectroscopy cell, T-format optics are required with simultaneous collection of vertical and horizontal emission paths for polarized measurements. This approach can lead to instrumental problems involving the matching of the photomultiplier responses of the two detection arms, or alternatively requires the use of optical fibers to transmit emission intensities from the high pressure spectroscopy cell via more conventional L-configuration optics.
It can be seen that there exists a need for methods that address the shortcomings of approaches discussed above. The present invention is directed to this important end.